Whole-mount in situ mRNA localization in developing ovules and seeds of Arabidopsis

The functional annotation of completely sequenced genomes requires fast and reliable procedures for the systematic determination of in situ patterns of gene expression. Although several whole-mount hybridization protocols have been successfully used to localize mRNAs in soft and easily dissected tissues such as roots, seedlings, and developing flowers, their use has been hampered by the small size and inaccessibility of developing ovules and megagametophytes. We have implemented a whole-mount in situ hybridization procedure that overcomes these difficulties in Arabidopsis thaliana. Multiple developing gynoecia, mature ovules, and nascent seeds are dissected, fixed, and embedded on a soft layer of 15% polyacrylamide before being processed for hybridization with digoxygenin-labeled RNA probes. We characterized the pattern of expression of a gene encoding a UDP-D-glucuronate 4-epimerase (GAE5) and demonstrated that the procedure can be used to determine highly dynamic patterns of mRNA localization early during development of ovules. We confirmed the reliability of our approach by detecting uidA (GUS) mRNA localization patterns in ovules from enhancer detector or transformant lines that showed glucuronidase (GUS) protein localization in specific cells of the megagametophyte. The method can be successfully used to localize mRNA transcripts and achieve single-cell resolution in both sporophytic and gametophytic cells at all stages of development of megagametophytes and during seed initiation. It constitutes an initial step toward the automation of high-throughput in situ hybridization procedures for functional studies of female reproductive development and early seed formation.